Throughout this application, various publications are referenced by author and date and by arabic numbers in superscript. Full citations for these publications may be found listed at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.
The development and differentiation of embryonic neurons culminates in synapse formation. Neuronal development is an intricate process that involves a cascade of inductive interactions between a neuron and the pre- and postsynaptic partners of that neuron. These highly regulated events are important for the establishment of reliable, yet plastic, synaptic formation and transmission. Correct expression of an array of transmitter-gated channels by neurons is clearly essential to synaptic differentiation, and yet the developmental regulation of this process is poorly understood. In fact, despite overwhelming advances in probing the molecular and biophysical details of ion channels gated by gamma-amino butyric acid (GABA), glycine, glutamate and acetylcholine (ACh) (Betz, 1990; Deneris et al., 1991; McGehee et al., 1995; Role, 1992; Sargent, 1993) the corresponding embryonic versions of these receptors have evaded analysis. Characterization of the biophysical properties of ligand-gated channels in developing neurons and description of their evolution to the mature receptor profile is limited (Brussard et al., 1994; Moss and Role, 1993; Margiotta and Gurantz, 1989). Furthermore, little is known about the mechanism of these changes.
The study of embryonic ligand-gated channels and subsequent modifications of their functional profile during neural development is difficult. Receptor expression prior to synaptogenesis is at a low level. Synapse formation is not synchronous. In the few cases studied, the developmental changes in receptor function are vast (Berg et al., 1989; Engisch and Fischbach, 1992; Arenella et al, 1993; Deneris et al, 1991; McGehee and Role, 1995; Role, 1992; Sargent, 1993). In the establishment of mature synapses, profound alterations in the expression profile of neuronal ligand-gated channels occur. In addition to these changes in expression levels, changes in the cellular distribution, the subunit composition and the biophysical and pharmacological properties occur as well (Margiotta and Gurantz, 1989; Moss and Role, 1993; Moss et al., 1989; Devay et al, 1994; Arenella et al, 1993; Jacob, 1991; Mandelzys et al, 1994; Smith et al, 1983; Vernallis et al, 1993). The interactions between presynaptic and target neurons may play a large role in the extrinsic influences which are believed to modify receptor function throughout development. The mechanism of receptor development remains unclear, however, presynaptic input, target cell regulation, synaptic activity or molecular signals independent of transmission may be involved.
Diversity of Neuronal Nicotinic Receptors
One important feature of neuronal ligand-gated channels, nicotinic acetylcholine receptors (nAChRs) in particular, is the diversity of component subunits and the resultant diversity in channel subtypes (Boulter et al, 1986; Conroy et al., 1992; Grynkiewicz et al., 1985; Lindstrom et al., 1990; Luetje and Patrick, 1991; McGehee and Role, 1995; Papke and Heinemann, 1991; Ramirez-Latorre et al., submitted; Role, 1992). Neuronal nAChRs were the first of the ligand-gated ion channels studied to display this degree of structural and functional complexity. Although nAChRs comprise only two distinct subunit types, there are multiple homologous forms of each subunit encoding gene. There are 8 neuronal “α” subunit genes (α1-α8) and 3 neuronal “β” subunit genes (β2-β4) cloned to date (Boulter et al., 1986; Heinemann et al., 1990; Nef et al., 1988; Seguela et al., 1993; Wada et al., 1989). With this array as a starting point, there could be more than 105 varieties of pentameric nAChR complexes (McGehee et al., 1995 and Role, 1992). Study of native nAChRs indicates that the actual number of subunit combinations is less than theory would predict. Biochemical, immunochemical, and antisense deletion experiments to identify native compositions of nAChRs demonstrate that relatively few subunit combinations are likely to be found in native nAChRs. For example, the nAChRs expressed by autonomic and habenula neurons have been studied in detail (Brussard et al., 1994; Devay et al., 1994; Listerud et al., 1991; Clarke et al., 1986) and provide specific examples of the subunit composition of each nAChR channel subtype expressed. In view of the documented evolution of these neuronal nAChR channels during embryonic development, and the array of molecular and biophysical tools available to study these channels in detail, an understanding of the developmental regulation of nAChR subunit and channel subtype diversity may be close at hand. Numerous studies implicate the interaction during the formation of synaptic connections between the presynaptic and postsynaptic cells in the development of mature neuronal receptors (Arenella et al., 1993; Boyd et al., 1988; Brussaard et al., 1994b; Brussaard et al., 1994; Devay (in preparation; Devay et al., 1994; Gardette, et al., 1991; Jacob 1991; Levey et al., 1994; Mandelzys et al., 1994; Moss et al., 1989).
Regulation of Neuronal Phenotype During Development: Contribution of Target Interactions.
Neuronal differentiation is induced by the interaction of developing neurons with target cells. One example is that of the evolution of transmitter phenotype in a special class of sympathetic neurons that evolve from an adrenergic to a cholinergic phenotype in the course of normal development. Although early on, these neurons synthesize, package and release catecholamines, the formation of synapses with the target sweat glands is accompanied by a change in transmitter expression that ultimately produces a mature cholinergic phenotype. This change in transmitter expression requires both pre- and postsynaptic signals. Thus, catecholamine release from the embryonic neuron is required to induce the release of a cell differentiation factor\leukemia inhibitory factor (CDF/LIF)-like factor called sweat gland factor (SGF) from the presumptive sweat glands. SGF, released via activation of target adrenergic receptors, interacts, with specific receptors on the innervating neuron. SGF induces the cellular machinery required for ACh synthesis and release in the presynaptic neuron. Thus, the attainment of a mature transmitter phenotype is regulated by both synaptic activity and target derived signals, offering an explanation for how the innervation of target tissues regulates nAChR expression in CNS and PNS neurons.
This example is one of many implicating target-derived factors in the control of neuronal survival, proliferation, differentiation, migration, and neurite outgrowth. Although there are many factors that could mediate target effects on neuronal differentiation, the expression patterns and biological activities of factors identified to date identify a few candidates for proposed studies of nAChR regulation. (1) Ciliatory neurotropic factor (CNTF) mimics the effect of SGF in inducing a cholinergic phenotype. It has also been shown to promote differentiation of sympathetic precursor cells and likely participates in target-induced changes in nAChR expression. CNTF is expressed in numerous sympathetic targets including smooth muscle and kidney. (2) Activin and related members of the Transforming Growth Factor β (TGFβ) family, can also regulate the differentiation of the transmitter phenotype in autonomic neurons. These factors are expressed in sympathetic targets such as smooth muscle, sweat glands etc. (3) CDF/LIF mimics SGF by inducing a cholinergic phenotype. This factor is secreted by smooth muscle and heart muscle. Although less is known about either the activity or the distribution of these factors in the central nervous system (CNS), it is likely that CNS and peripheral nervous system (PNS) neurons may be regulated by similar signaling molecules.
Regulation of Receptor Synthesis and Distribution in Muscle: Contribution of Presynaptic Signals
Classical studies of Fischbach, Cohen, and McMahan of the nerve-muscle junction demonstrate that the incoming motor nerve is a potent regulator of muscle-nAChRs. Prior to innervation, muscle expresses an embryonic form of nAChR which is diffusely distributed over the cell surface. The expression of the muscle-nAChR is eventually downregulated to a diffuse distribution. Elimination of muscle-nAChRs by innervation is accompanied by an increase in local synthesis, insertion and formation of high-density clusters of muscle-nAChR at the synaptic site. At later stages of synaptic development, there are marked changes in the biological properties of muscle-nAChR channels due to alterations in subunit gene expression. This produces “adult” type muscle-nAChR complexes of distinct subunit composition. Molecular signals that are believed to mediate these changes in muscle-nAChR distribution and synthesis have been identified and cloned, namely, agrin and AChR Inducing Activity (ARIA). Recombinant agrin alters the distribution of pre-existent muscle-nAChRs with no effect on synthesis or insertion of new receptors. In contrast, recombinant ARIA induces muscle-nAChR subunit gene expression, increasing the rate of appearance of new surface receptors from 3-5%/hr to 10-20%/hr.
It is possible that there are common regulatory mechanisms between nAChR and muscle-nAChR. It is believed that nAChRs on both CNS and PNS neurons evolve from low density and diffuse distribution to clustered and highly dense synaptic patches following innervation. Finally, like muscle-nAChRs, there are marked changes in the biophysical properties of nAChRs during development and presynaptic input may induce some of these changes, e.g., channel conductance and opening frequency.
Despite the essential role of ligand-gated ion channels in synaptic transmission between neurons, little is known about changes in their expression, function, distribution and subunit composition during neural development. Nicotine-induced enhancement of acquisition and consolidation of short term memories is believed to be mediated by presynaptic nAChR activation since this activation facilitates a broad array of CNS synapses. In view of the impact that developmental changes in nAChRs have on neuronal excitability, synaptic efficacy and synaptic plasticity, studies of the regulatory controls of nAChR expression are essential. One avenue of study focuses upon the proteinaceous factors that appear to modulate receptor gene expression. One factor previously identified and cloned is heregulin (See Vandlen and Holmes, U.S. Pat. No. 5,367,060).
To date, 10 different proteins that result from alternative splicing of the heregulin gene have been described. Among these are the growth factors neu differentiation factor (NDF) (Wen et al., 1992), glial growth factor (GGF) (Marchionni et al., 1993), ARIA (Falls et al, 1993; Fischbach et al., 1994), and the heregulin isoforms (Holmes, 1992; Wen et al., 1994). The reported isoforms are principally membrane bound proteins which can be solubilized by proteolysis. The extracellular domains of these proteins consist of an N-terminal domain followed by an immunoglobulin-like (Ig-like) domain, a linker region, an EGF-like domain, and a second linker region (FIG. 5). These proteins are ligands for the epidermal growth factor (EGF) family of receptor tyrosine kinases. Binding of the ligand to the EGF receptor family members erbB3/HER3 or HER4 results in activation of the tyrosine kinase activity of the receptor. Other family members can be activated by trans-phosphorylation via the activated members.
Other members of the heregulin/NDF/ARIA family have been described in previous patent publications. PCT International Publication No. WO 94/08007, published Apr. 14, 1994, entitled “Trophic factor having ion channel-inducing activity in neuronal cells” describes neurotrophic factors designated as ARIA, which are able to induce the formation of ion channels. This publication also shows how ARIA is associated with both nervous tissue and skeletal muscle. ARIA has an Ig-like domain and an EGF-like domain in the extracellular region. U.S. Pat. No. 5,367,060, issued Nov. 22, 1994, entitled “Structure, production and use of heregulin”, from U.S. Ser. No. 847,743, filed Mar. 6, 1992 by Richard L. Vandlen and William E. Holmes, discloses a polypeptide with a binding affinity for the p185HER2 receptor. Vandlen and Holmes also disclose purification methods required to isolate heregulin and uses of the heregulins and antibodies specific to the heregulins as therapeutic agents. This polypeptide is related to but distinct from the ARIA protein.